Although many bacteria are harmless or beneficial, some bacteria are pathogenic and can cause disease. Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, kills about 2 million people a year worldwide. Pathogenic bacteria contribute to other important diseases, such as pneumonia, which can be caused by bacteria such as Streptococcus and Pseudomonas bacteria, and food borne illnesses, which can be caused by bacteria such as Shigella, Campylobacter and Salmonella. Other disease-causing bacteria include Bacillus anthracis (anthrax), Clostridum tetani (tetanus), Corynebacterium diphtheriae (diphtheria), Helicobacter pylori (stomach ulcers), Legionella pneumophila (Legionnaire's disease), Mycobacterium leprae (leprosy), Salmonella typhi (typhoid fever), Staphylococcus aureus (sepsis), Vibrio cholerae (cholera) and Yersinia Pestis (bubonic plague). While the availability of antibiotics has rendered many bacterial diseases treatable, the emergence of antibiotic-resistant strains of bacteria has presented new challenges. There is therefore an ongoing need for the development of new analytical and diagnostic methods for studying and detecting bacteria.
Bacteria are often classified into Gram-positive and Gram-negative strains by their visual staining properties using crystal violet, a triarylmethane dye. The Gram staining method is a common tool for detecting and differentiating bacteria. Gram stains are commonly used for clinical diagnostic purposes, identification of a bacterial organism, as well as detecting them in environmental samples. The procedure involves staining bacterial samples with crystal violet (FIG. 1A), which binds to the peptidoglycan layer of Gram-positive and negative bacteria (FIG. 1B). As shown in FIG. 1B, Gram-positive and Gram-negative bacteria differ in the structure of their cell wall. Gram-positive bacteria have a thick peptidoglycan layer whereas Gram negative bacteria only have a thin peptidoglycan layer covered by lipopolysaccharides and lipoproteins. Subsequent treatment with iodine solution results in formation of an insoluble complex with crystal violet to form. Upon decolorization with alcohol or acetone, only Gram positive bacteria remain purple, while Gram-negatives lose the purple color. Beveridge, Biotech. Histochem., 2001, 76, 111-8; Bartholomew et al., Bacteriol. Rev., 1952, 16, 1-29; Bottone, Lab. Med., 1988, 19, 288-91. Despite the simplicity and robustness of the staining procedure, the final detection still relies on optical microscopy which is user dependent and therefore not entirely error free.
A number of antibiotics have been developed to treat Gram-positive infections, many of which work either by inhibiting cell wall synthesis or by blocking transcription/translation processes. Vancomycin is a commonly used glycopeptide antibiotic, whose action primarily results in inhibition of cell wall synthesis. Specifically, vancomycin exerts its antibacterial activity by forming hydrogen bonds with the terminal D-alanyl-D-alanine (D-Ala-D-Ala) moieties of the N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) peptide subunits. Nagarajan et al. Antimicrob. Agents Chemother. 1991, 35, 605-609; Reynolds, Eur. J. Clin. Microbiol. Infect. Dis. 1989, 8, 943-950. This binding prevents incorporation of the NAM/NAG-peptide subunits into the major structural component of Gram-positive cell walls, the peptidoglycan matrix, and thus results in inhibition of cell wall synthesis and ultimately bacterial cell death. The increasing prevalence of vancomycin-resistant organisms, however, have now led to the development of newer generation antibiotics including daptomycin, linezolide and pristinamycin. Daptomycin binds to the cell wall of Gram-positive bacteria via its hydrophobic tail, resulting in perturbation and depolarization of the cell membrane. Steenbergen, et al., J. Antimicrob. Chemother. 2005, 55, 283-288.
Trehalose, also known as mycose or tremalose, is a natural alpha-linked disaccharide formed by an α,α-1,1-glucoside bond between two α-glucose units. Trehalose is present as a free disaccharide in the cytoplasm of mycobacteria and as a component of cell-wall glycolipids implicated in tissue damage associated with mycobacterial infection, and is synthesized by Mycobacteria through three pathways. De Smet et al., Microbiology, 2000, 146, 199-208. Trehalose is found in the outer portion of the mycobacterial cell envelope along with the glycolipids trehalose dimycolate (TDM) and trehalose monomycolate (TMM). Hoffmann et al., Proc. Natl. Acad. Sci. USA. 2008, 105, 3963-3967. Uptake of unnatural trehalose analogs has been described as a reporter for mycobacteria such as M. Tuberculosis. Backus et al., Nat. Chem. Biol., 2011, 7(4), 228-235; WO2011/030160.
Magnetic particles have been studied and used for a number of biomedical applications.
Preparations of magnetic particles designed for separation and extraction use particles that are amenable to easy manipulation by weak applied magnetic fields. These materials are typically micron sized and have a high magnetic moment per particle; their effects on water relaxation rate are unspecified and not relevant to their application. Smaller particles, in contrast, such as nanoparticles do not respond to the weak, magnetic fields of hand held magnets.
Magnetic nanoparticles are a class of nanoparticle which consist of magnetic elements such as iron, nickel and cobalt and their chemical compounds and can be manipulated using a magnetic field. A number of different approaches to preparing such particles have been described. Lu et al., Angew. Chem. Int. Ed. Engl., 2007, 46, 1222-1244.
Magnetic nanoparticles are typically smaller than 1 μm in diameter (typically 5-500 nm), while larger microbeads can be, e.g., from 0.5-500 μm in diameter. In many of the applications of magnetic nanoparticles, the particles perform best when the size of the nanoparticles is below a critical value, which is dependent on the material but is typically around 10-20 nm, when each nanoparticle becomes a single magnetic domain and shows superparamagnetic behavior when the temperature is above a particular temperature (a blocking temperature). In a supermagnetic state, nanoparticles are sufficiently small that their magnetization can randomly flip direction so that in the absence of a magnetic field the average magnetization can appear to be zero. An external magnetic field can magnetize the nanoparticles, similarly to a paramagnet. However, the magnetic susceptibility of supermagnetic nanoparticles is much greater than that of paramagnet.
Magnetic nanoparticles have been studied, in particular, for potential biomedical applications, including magnetic resonance imaging contrast enhancement, tissue repair, immunoassay, detoxification of biological fluids, hyperthermia, drug delivery and in cell separation. Gupta et al., Biomaterials, 2005, 26(18), 3995-4021.